Products and Method for the Decontamination of Prions

ABSTRACT

The invention relates to the use of Cu and the derivatives thereof, for the decontamination of prions, especially for decontaminating medical devices or medical surgical devices at risk, for decontaminating work surfaces, and for decontaminating any potentially infectious compound.

The invention relates to products and methods for the decontamination ofproducts and equipment infected with unconventional transmissible agents(UTA) responsible for transmissible subacute spongiformencephalopathies.

More specifically, the invention concerns the decontamination ofproducts and equipment infected with prions which accumulate mainly inthe brain of the host, more particularly with an abnormal isoform,PrP^(sc) (standing for PrP^(scrapie)) which results from aconformational modification of a protein PrP^(c) (cellular PrP) encodedby the host.

The appearance of a new variant of Creutzfeldt-Jakob disease in GreatBritain and its possible link with bovine spongiform encephalopathyraised the possibility of the contagiousness of the disease, inparticular via infected products of animal origin, especially via foodsand via contaminated medical or surgical equipment.

In fact, the current techniques for disinfection and sterilization donot make it possible easily to destroy prions, which are resistant tothe majority of the methods normally used. Administrative directiveshave laid down the rules to be followed in this regard, but they arevery awkward to implement and onerous and they necessitate the use oftoxic substances.

In the light of this situation, the inventors sought compounds enablingsimple and effective decontamination from the pathological agent, usableon a great majority of surfaces and medical and surgical equipment.Their studies have demonstrated the efficacy of certain metalderivatives in this regard.

The purpose of the invention is thus the use of such compounds for priondecontamination.

It also concerns a method for the treatment of infected equipment andproducts comprising the use of these compounds.

The invention also concerns the use of Cu and of derivatives thereof,for prion decontamination.

According to a supplementary provision of the invention, thesederivatives are used with H₂O₂.

Preferably, the metal derivative is CuSO₄.

The compound or compounds used according to the invention areadvantageously in the form of aqueous solutions.

The study of the action of these derivatives on the decontamination fromthe pathogenic agents responsible for prion diseases demonstrated theirgreat efficacy for decontaminating infected products or equipment.

Advantageously, these are moreover non-toxic, biodegradable and readilymanipulable compounds.

The invention thus equally concerns a process for decontamination ofproducts or equipment infected with pathogenic agents responsible forprion diseases, characterized in that it comprises placing them incontact with at least one compound such as defined above or with asolution containing it, and if necessary with H₂O₂.

In a preferred mode of implementation of the invention, the metalderivative is CuSO₄.

The decontamination treatment is preferably carried out with a solutioncontaining said compound at a level of at least 500 μM, especially from500 to 1000 μM.

H₂O₂, when it is used, is present in the solutions at a level of about50 mM.

Satisfactory results are obtained by operating at ambient temperature,for a period of the order of 15 to 60 mins, in particular for about 30mins.

This compound or these compounds and solutions thereof thus are of greatinterest in the context of hospital use, where they enable, inparticular, decontamination of medical or medical-surgical devices atrisk, such as multiple use equipment, such as endoscopes, probes(dialysis), or also decontamination of work surfaces, such as a drainingboard or floor, also at risk.

Said compounds or solutions thereof are also used in particular fordecontaminating for example an infectious material of cerebral originand biological products originating from subjects who are carriers ofinfectious forms of Creutzfeldt-Jakob disease. The invention alsoconcerns the use of said compounds and of solutions thereof fordecontaminating products originating from the blood or biologicalmaterial used in transplants.

Advantageously, these compounds and solutions have been found to beeffective on the prion strain linked with BSE (transmissible bovinespongiform encephalitis or mad cow disease). They are applicable to alltypes of prion whatever the strain and origin of the agent may be.

They are also advantageously usable in the context of anagricultural/food application, especially for the decontamination of anypotentially infectious compound and in particular animal meal or othercontaminated products of animal origin. Applications of interest alsoinclude the decontamination of premises, such as abattoirs, with areasand equipment at risk of being in contact with the infectious agents. Inparticular, those deriving from ruminants may be mentioned.

Other characteristics and advantages of the invention are given in theexamples that follow, wherein reference is made to FIGS. 1 and 2, whichrepresent, respectively:

EXAMPLE 1 Prion Decontamination Solution Containing CuSO₄ and H₂O₂

a) Study of the action of copper associated with H₂O₂ on the degradationof the PrP^(sc) present in infectious brain homogenates of mice.

Solutions of CuSO₄ and of H₂O₂ at different concentrations are added tosamples consisting of infectious extracts of murine brain homogenatesand are left in contact for about 30 mins at ambient temperature.

The samples are then deposited on a polyacrylamide SDS-PAGE gel afteradjustment of the protein concentrations and digestion with proteinase K(PK) at a concentration of 1 mg of PK per 50 mg of protein. The presenceof PrP^(sc) is revealed by Western blotting.

The results obtained are illustrated by FIG. 1. It can be seen that at aconcentration of 100 μM of CuSO₄ and 50 mM of H₂O₂, the infectioussamples display decreased levels of PrP^(sc). At a concentration of 500μM of CuSO₄ and 50 mM of H₂O₂, the level of PrP^(sc) present in theinfectious homogenates becomes undetectable in Western blotting (tracks7 and 8). At this concentration, the effect is potentiated by the actionof H₂O₂.

With the use of higher doses of CuSO₄ from 1 mM to 10 mM, the PrP^(sc)signal can be made to disappear with copper alone and H₂O₂ is no longernecessary.

These results are confirmed on repeating these experiments onhomogenates deriving from the brains of mice infected with the 22 Lprion strain. Similar results have been obtained on homogenates derivingfrom the murine Chandler strain and BSE, which shows that thedecontaminating effect is not strain-dependent.

b) Study of the infectivity of infectious brain homogenates treated witha high concentration of copper in vitro.

22 L brain homogenates were treated for about 30 mins with differentconcentrations of CuSO₄ with and without H₂O₂ at differentconcentrations.

These homogenates are dialyzed, then deposited onto cells of murineneuroblastomas having the capacity to replicate the infectious agent.

Untreated brain homogenates are used as the control.

A Western blot is performed after 6 passages on the cell lysates andafter digestion with PK, in order to test for the presence of PrP^(sc)signifying that the samples tested still remain infectious.

The results obtained are given in FIG. 2. It can be seen that theinfection is decreased by the treatment.

EXAMPLE 2 Tests In Vivo

A confirmation of these results observed in vitro has been effected.Infectious 22 L brain homogenates were treated in particular with 500 μMof CuSO₄ and 100 mM of H₂O₂. The homogenates, treated or untreated, wereinoculated into mice by the intracerebral route. The control animalsinoculated with the untreated homogenates all fell ill in 168 days±2days. Certain animals inoculated with the treated homogenates are stillalive after more than 300 days. This result demonstrates that areduction of at least 7 powers of ten in the infectious titer can beobtained by this decontamination method.

EXAMPLE 3 Decontamination of Surgical Equipment

Equipment such as endoscopes is immersed in a solution containing 1 mgof CuSO₄ for 20 mins. The tests performed to identify the presence ofprions after this treatment were found to be negative.

EXAMPLE 4 Study of the Efficacy of Decontamination

The data concerning the efficacy of decontamination tested in vivo afterinoculation into the mouse are given below. For this, control anddecontaminated brain homogenates were intracerebrally inoculated intothe mouse (C57B1).

The survival times of the mice are as follows:

1) Inoculation of control infectious homogenates: 167.6±0.5 days.

2) Homogenates treated with CuSO₄ (0.5 mM, 30 mins, ambienttemperature): 200.2±8.9 days.

3) Homogenates treated with CuSO₄ (1 mM, 30 mins, ambient temperature):217.2±13.4 days.

4) Homogenates treated with CuSO₄ (1 mM)+H₂O₂ (100 mM, 30 mins, ambienttemperature): 266.4±15.6 days.

In the light of these incubation periods under these differentconditions and a titration curve for the infectious agent effected onthese mice, it can be estimated that the decontamination obtained isgreater than 10⁴ in the homogenates treated just with CuSO₄ and greaterthan 10⁵ in the homogenate treated with CuSO₄+H₂O₂.

1. The use of derivatives of Cu and of its derivatives for the priondecontamination of infected products and equipment.
 2. The use asclaimed in claim 1, characterized in that these derivatives are usedwith H₂O₂.
 3. The use as claimed in claim 1, characterized in that themetal derivative is CuSO₄.
 4. The use as claimed in claim 1,characterized in that the compound or compounds used are in the form ofaqueous solutions.
 5. A method for decontamination of products orequipment infected with pathogenic agents responsible for priondiseases, characterized in that it comprises placing them in contactwith at least one compound such as defined in claim 1, or a solutioncontaining it, and if necessary also with H₂O₂.
 6. The method as claimedin claim 5, characterized in that the metal derivative is CuSO₄.
 7. Themethod as claimed in claim 5, characterized in that the decontaminationtreatment is carried out with a solution containing said compound at alevel of at least 500 μM, in particular from 500 to 1000 μM.
 8. Themethod as claimed in claim 5, characterized in that H₂O₂ is present inthe solutions at a level of about 50 mM.
 9. An application of the methodas claimed in claim 5 for the decontamination of medical ormedical-surgical devices at risk, such as multiple use equipment, suchas endoscopes, or also decontamination of work surfaces, such as adraining board or floor.
 10. The application of the method as claimed inclaim 5 for the decontamination of any potentially infectious compoundand in particular animal meal or other contaminated products of animalorigin.